Development and evaluation of a high-throughput, low-cost genotyping platform based on oligonucleotide microarrays in rice

Abstract

Background: We report the development of a microarray platform for rapid and cost-effective genetic mapping, and its evaluation using rice as a model. In contrast to methods employing whole-genome tiling microarrays for genotyping, our method is based on low-cost spotted microarray production, focusing only on known polymorphic features.

Results: We have produced a genotyping microarray for rice, comprising 880 single feature polymorphism (SFP) elements derived from insertions/deletions identified by aligning genomic sequences of the japonica cultivar Nipponbare and the indica cultivar 93-11. The SFPs were experimentally verified by hybridization with labeled genomic DNA prepared from the two cultivars. Using the genotyping microarrays, we found high levels of polymorphism across diverse rice accessions, and were able to classify all five subpopulations of rice with high bootstrap support. The microarrays were used for mapping of a gene conferring resistance to Magnaporthe grisea, the causative organism of rice blast disease, by quantitative genotyping of samples from a recombinant inbred line population pooled by phenotype.

Conclusion: We anticipate this microarray-based genotyping platform, based on its low cost-per-sample, to be particularly useful in applications requiring whole-genome molecular marker coverage across large numbers of individuals.

Figure 1

Positions of SFP markers on the rice pseudomolecule map. SFPs with oligonucleotide sequences complementary to Nipponbare are shown as black and those complementary to 93-11 are shown in grey. SFPs confirmed by PCR are shown in blue (Nipponbare) and red (93-11). The positions of the centromeres are indicated with an "X".

Figure 2

A volcano plot comparing Nipponbare and 93-11 genomic DNA hybridizations with the 880 SFPs. SFPs with oligonucleotide sequences complementary to Nipponbare are shown as black and those complementary to 93-11 are shown as grey.

Figure 3

Neighbor Joining tree using the SFP marker data to show the genetic relationships between the five sub-populations of rice. Neighbor joining trees were constructed using four accessions per subpopulation. The bootstrap values are out of 10,000.

Figure 4

Site-by-site probabilities for the population of origin of alleles across the twelve chromosomes of each accession. There are four accessions per sub-population plus Nipponbare and 93-11 reference sequences. Calculations are done using the linkage model of STRUCTURE.

Figure 5

Significance of SFP fold change measurements between pools of DNA from blast resistant and susceptible RILs. SFP markers showing association of the resistant parent (SHZ) allele with the resistant pool are assigned positive values and association with the susceptible parent allele (LTH) with the resistant pool are assigned negative values. The line is drawn using loess smoothing.

Figure 6

Location of the five most significant SFP markers (ordered A-E) associated with blast resistance in the bulked segregant experiment. The SFP markers are co-located with the previously mapped microsatellite marker (RM179), known to be linked to blast resistance.